Method for creating perfusable microvessel systems

ABSTRACT

A method for the creation of endothelial parent vessels from human vascular endothelial cells in vitro in a culture perfusion device (CPD) including a collagen chamber, inlet ports, a capillary tube, and an outlet port. A collagen solution is injected into the collagen chamber through a syringe needle until the chamber is filled with collagen. The CPD is perfused by filling the inlet ports and sequentially priming the inlet ports, and the outlet ports. A perfusable channel is created in the collagen chamber and a concentrated suspension of endothelial cells is injected into the inlet ports. The endothelial cells are injected into the at least one perfusable channel and incubated to attach to the walls of the perfusable channel. The cells are distributed within the CPD; and perfused to form a parent vessel having homogeneous monolayers of cells.

RELATED APPLICATIONS

This application claims priority from and is a divisional application ofU.S. application Ser. No. 11/860,471, now U.S. Pat. No. 8,003,388, ofNeumann, filed Sep. 24, 2007, entitled “Method for Creating PerfusableMicrovessel Systems,” which, in turn is a continuation-in-part of U.S.application Ser. No. 11/388,920, now U.S. Pat. No. 7,622,298, ofNeumann, filed Mar. 24, 2006, entitled “Method for Creating PerfusableMicrovessel Systems.” Both of these applications are Hereby incorporatedby reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. 1 R21HL081152-01 awarded by NIH National Heart, Lung, and Blood Institute.The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for the study of physiologicaland pathological vascular growth, and vascular growth in response toangiogenic or angiostatic factors.

TECHNICAL BACKGROUND

During normal processes of vascular growth (e.g., the menstrual cycle,placentation, changes in adiposity, wound repair, inflammation), thecreation of new blood vessels is regulated and eventually ceases.Significantly, the deregulation of vascular growth is a critical elementof pathology. For example, tumor growth, diabetic retinopathies,arthritis, and psoriasis involve excessive proliferation of bloodvessels that contributes directly to the pathological state. Incontrast, impairment of vascular growth, characteristic of agedindividuals, compromises the healing of wounds and the revascularizationof tissues rendered ischemic by trauma or disease. Therefore, anunderstanding of the mechanisms that direct the assembly new bloodvessels, and the processes that start and stop vascular growth, arecentral to the development of strategies to control vascularization indisease.

During the growth of new blood vessels (angiogenesis), sprouts arisefrom endothelial cells that line the lumens of capillaries andpostcapillary venules—the smallest branches of the vascular system.Angiogenesis is a complex, multi-step process. Although publishedstudies of angiogenesis number in the many thousands, the cellularmechanisms that mediate and regulate angiogenic growth and morphogenesisare poorly understood.

The details of angiogenic sprouting are difficult to observe in“real-time” in vivo because of the opacity of most tissues. Tissuesections are difficult to reconstruct in 3D and do not communicate thedynamic nature of vascular growth. Moreover, the region near the tips ofangiogenic sprouts—a critical area of control of vascular invasion andmorphogenesis—is rarely found in tissue sections. In order to overcomethe limitations of conventional histology, a variety of “models” ofangiogenesis in vivo and in vitro have been developed.

Models of angiogenesis in vivo: To circumvent the opacity of livingtissues, investigators have observed angiogenesis through “windows” inliving animals that include the naturally transparent tails of amphibianlarvae (Clark and Clark 1939), or specialized viewing chambers eitherimplanted into rabbit ears (Clark and Clark 1939), mouse skin (Algire,Chalkley et al. 1945) and hamster cheek pouches (Greenblatt and Shubi1968) or developed from rabbit corneal pockets (Gimbrone, Cotran et al.1974) or chick chorioallantoic membranes (Ausprunk, Knighton et al.1974). From these early, largely descriptive studies came validation ofthe central paradigm of tumor-induced vascular chemotaxis and thecorresponding discovery of diffusible, tumor-derived molecules thatpromote vascular growth. Newer assays of angiogenesis in vivo measurevascular ingrowth into polymeric sponges or plugs of gelled basementmembrane proteins implanted subcutaneously into rodents (Passaniti,Taylor et al. 1992; Andrade, Machado et al. 1997; Akhtar, Dickerson etal. 2002; Koike, Vernon et al. 2003). For all of their elegance,approaches in vivo are made difficult by: (1) intra-species variation inangiogenic response from animal to animal; (2) the lack of translationof results from one species to another; (3) high costs of animalpurchase and maintenance; (4) public disapproval of the use of animalsfor research purposes; and (5) complexities encountered in animalsurgeries and in the visualization and evaluation of results.Two-dimensional (2D) models of angiogenesis in vitro: In an effort tounderstand the molecular mechanics of angiogenesis, endothelial cellsisolated from large vessels were cultured in flat dishes until theyformed confluent, pavement-like monolayers that simulated theendothelial linings of blood vessels (Jaffe, Nachman et al. 1973;Gimbrone 1976). Although useful as models of proliferative responses toendothelial injury in large blood vessels (Gimbrone, Cotran et al. 1974;Fishman, Ryan et al. 1975; Madri and Stenn 1982; Madri and Pratt 1986;Jozaki, Marucha et al. 1990; Rosen, Meromsky et al. 1990), monolayercultures of endothelial cells on rigid substrata do not typicallyorganize into capillary-like tubes in simulation of angiogenesis. In1980, however, following successful long-term culture of capillaryendothelial cells (Folkman, Haudenschild et al. 1979), it was reportedthat 20-40 day cultures of bovine or human capillary endothelial cellsdeveloped a 2D cellular network on top of the confluent cellularmonolayer, a process termed “angiogenesis in vitro” (Folkman andHaudenschild 1980). The endothelial cells of the network appeared as“tubes” with “lumens” filled with a fibrillar/amorphous material thatwas interpreted to be an endogenously-synthesized network of “mandrels”on which the cells organized. Later studies reported similar 2D networkformation by endothelial cells from large vessels (Maciag, Kadish et al.1982; Madri 1982; Feder, Marasa et al. 1983) and by endothelial cellsseeded on top of malleable, hydrated gels of basement membrane proteins(e.g. Matrigel® gel)(Kubota, Kleinman et al. 1988).

Although 2D models of vascular development remain in use today (theMatrigel®-based assay (Kubota, Kleinman et al. 1988) is availablecommercially), such models lack the following 5 defining characteristicsof true angiogenesis:

-   1. Invasion—Endothelial cells in 2D models form networks on top of    extracellular matrix and show little propensity to burrow into the    extracellular matrix (Vernon, Angello et al. 1992; Vernon, Lara et    al. 1995).-   2. Directionality—In 2D models, the networks of endothelial cells    form in vitro more or less simultaneously throughout a field of    pre-positioned cells, whereas angiogenesis in vivo involves the    vectorial invasion of extracellular matrix by filamentous sprouts    that arborize by multiple levels of branching.-   3. Correct polarity—Although the 2D models make unicellular tubes    that markedly resemble capillaries (Maciag, Kadish et al. 1982;    Feder, Marasa et al. 1983; Sage and Vernon 1994), their polarity is    “inside-out”, that is, they deposit basement membrane material on    their luminal surfaces and have their thrombogenic surfaces facing    outward to the surrounding culture media (Maciag, Kadish et al.    1982; Feder, Marasa et al. 1983)—opposite to the situation in vivo.-   4. Lumen formation—Evidence that 2D models generate endothelial cell    (EC) tubes with patent lumens is weak. Typically, the endothelial    cell tubes have “luminal” spaces that are filled with extracellular    matrix (either exogenous or synthesized by the cells) (Maciag,    Kadish et al. 1982; Madri 1982; Feder, Marasa et al. 1983; Sage and    Vernon 1994; Vernon, Lara et al. 1995). Where present, patent lumens    usually appear as slit-like or narrow cylindrical spaces bounded by    thick walls of endothelial cell cytoplasm—quite different from the    inflated, thin-walled endothelial cell tubes that typify capillaries    in vivo.-   5. Cell specificity—The cellular networks in 2D models are generated    by mechanical processes that may be accomplished by non-EC cell    types (Vernon, Angello et al. 1992; Vernon, Lara et al. 1995).    Indeed, mathematical modeling has shown that any adherent cell type    capable of applying tensile forces to malleable, 2D extracellular    matrix (either synthesized endogenously or supplied (e.g., Matrigel®    gel)) can generate networks under optimal conditions (Manoussaki,    Lubkin et al. 1996).    Three-dimensional (3D) models of angiogenesis in vitro: The    recognition that angiogenesis in vivo occurs within a 3D    extracellular matrix has led to a variety of models in which    sprouting is induced within 3D gels of extracellular matrix in    vitro. In an early 3D model, endothelial cells dispersed within    collagen gels (Montesano, Orci et al. 1983) formed networks of cords    and tubes (Elsdale and Bard 1972). Although the endothelial cell    tubes exhibited correct polarity, the characteristics of invasion    and directionality were lacking (the endothelial cells were    pre-embedded and evenly dispersed in the extracellular matrix).    Nonetheless, this approach has proven useful in studies of lumen    formation (Davis and Camarillo 1996) and of responses of endothelial    cells to growth factors (Madri, Pratt et al. 1988; Merwin, Anderson    et al. 1990; Kuzuya and Kinsella 1994; Marx, Perlmutter et al. 1994;    Davis and Camarillo 1996).

In an alternative approach, 1 mm sections (rings) of rat aorta embeddedin a 3D plasma clot generated branching, anastomosing tubes (Nicosia,Tchao et al. 1982). Sprouts from the aortic rings exhibitedangiogenesis-like invasion and directionality in addition to polarity.Explant models utilizing aortic rings from rats or microvascularsegments from mice have been used to study the influence of tumors,growth factors, various extracellular matrix supports, and conditions ofaging on angiogenesis (Nicosia, Tchao et al. 1983; Mori, Sadahira et al.1988; Nicosia and Ottinetti 1990; Nicosia, Bonanno et al. 1992;Villaschi and Nicosia 1993; Nicosia, Bonanno et al. 1994; Nicosia,Nicosia et al. 1994; Nicosia and Tuszynski 1994; Hoying, Boswell et al.1996; Arthur, Vernon et al. 1998).

A variety of models exist that induce purified endothelial cells (asmonolayers or aggregates) to sprout invasively into underlying orsurrounding 3D extracellular matrix gels (Montesano and Orci 1985;Pepper, Montesano et al. 1991; Montesano, Pepper et al. 1993; Nehls andDrenckhahn 1995; Nehls and Herrmann 1996; Vernon and Sage 1999; Vernonand Gooden 2002). Each of these models has specific limitations thatinclude difficulty in visualizing sprout formation, limited sprouting, arequirement for sectioning, or lack of effectiveness with certain typesof endothelial cells.

Wolverine and Gulec have disclosed a 3D angiogenesis system (US2002/0150879 A1) that involves embedding a fragment of tumor tissue intoa matrix. The outgrowth of microvessels can be characterized to assaythe angiogenic potential of the tissue. However, this approach does notprovide luminal perfusion of the microvessels.

Neumann (the inventor here) et al. 2003, has disclosed the possibilityof creating perfused microvessels in vitro that can be included in anartificial tissue. Neumann et al. 2003 teaches using 127 micrometernylon fishing line as mandrels held by shrink tubing for makingmicrovessels. The vessels were made from rat aortic smooth muscle cellsembedded in agar. These microvessels were of an exploratory nature andnot suitable for creating a human vessel graft.

Two-dimensional models of vascular growth in vitro do not establish thedefining characteristics of angiogenesis listed previously, whereasexisting 3D models reproduce some or most of the characteristics.Importantly, none of the 3D models currently available reconstruct aparent blood vessel that contains a pressurized, flowing, circulatoryfluid. Consequently, none of the existing in vitro 3D models permitstudy of the contribution of luminal pressure and flow to vasculargrowth and morphogenesis.

SUMMARY OF THE DISCLOSURE

A method for creating networks of perfusable microvessels in vitro, isdisclosed. A mandrel is drawn through a matrix to form a channel throughthe matrix. Cells are injected into the channel, the channel having aninner wall. The matrix is incubated to allow the cells to attach to theinner wall. The channel is perfused to remove unattached cells to createa parent vessel, where the parent vessel includes a perfusable hollowchannel lined with cells in the matrix. The parent vessel is induced tocreate sprouts into the surrounding matrix gel so as to form amicrovessel network. The microvessel network is subjected to luminalperfusion through the parent vessel.

The present disclosure provides methods and systems that overcome thelimitations of existing models of angiogenesis by combining provenmethods for generating invasive, tubular, microvascular sprouts in 3Dextracellular matrix (ECM) with novel methodologies for the fabricationof a tissue-engineered parent vessel that will be the source of luminalflow. Via the perfusate, angiogenesis-modulatory compounds can beadministered to the luminal surface of endothelial cells where specifictarget receptors are known to reside. The presence of a luminal flow ofnutrient medium will substantially increase the survival time ofcapillary tubes in vitro. The disclosed angiogenesis system can be usedevaluate a variety of experimental parameters that includehypoxia/hyperoxia, test of specific soluble bioactive compounds, use ofgenetically modified cells, and gene delivery via viral transfection.The system allows the study of angiogenesis relative to wound repair,aging, cancer, and atherosclerosis. Importantly, a model following theteachings of the present invention may be adapted to provide fullyfunctional vascular systems capable of being incorporated intobioengineered artificial tissues.

The present disclosure also provides new and novel approaches, includinga manifold design for making microvessels, making microvessels fromendothelial cells and making larger vessels (e.g. having the size ofcoronary arteries). These and other important new teachings, including,for example, a method for creation of microvascular networks are evidentfrom the specification and claims hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B and FIG. 1C schematically show an example ofparent-vessel creation.

FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D schematically show an example of aknown heat-shrink process.

FIG. 3A schematically shows a known design for mountingculture/perfusion devices.

FIG. 3B schematically shows a design used in a manufacturing method formounting culture/perfusion devices.

FIG. 4A and FIG. 4B schematically show creation of manifolds forculture/perfusion devices.

FIG. 5A, FIG. 5B and FIG. 5C schematically show an alternative designfor microfabricated culture/perfusion devices.

FIG. 6 schematically shows a cell-seeding procedure.

FIG. 7 shows a schematic of a capillary network between twobioartificial parent vessels.

FIG. 8a shows an in vitro image of an example of a plurality of mandrelsafter seeding with smooth muscle cells.

FIG. 8b shows an example of a perfused muscle plate.

FIG. 9 schematically shows an alternate embodiment of a CPD.

FIG. 10 shows a single parent vessel growing sprouts into thesurrounding matrix.

FIG. 11 shows one parent vessel connected through a network of sproutsto a second parent vessel.

FIG. 12, an alternate method for creating parent cells by seeding cellsinto channels in a collagen matrix is shown

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The examples presented herein are for the purpose of furthering anunderstanding of the invention. The examples are illustrative and theinvention is not limited to the example embodiments. The method of thepresent invention is useful for the study of physiological andpathological vascular growth, and vascular growth in response toangiogenic or angiostatic factors. Other useful applications are tomethods that evaluate the angiogenic potential of cancer tissues and theresponse to antiangiogenic drugs. Additionally, the method of theinvention may be used to construct various wound-healing devices and forvascularization of tissue-engineered constructs.

In one example a method and device for the creation of perfusablethree-dimensional microvessel networks is disclosed. As used herein “EC”refers to endothelial cells, “SMC” refers to smooth muscle cells and“CAS” refers to coronary-artery substitutes.

Generally, the devices for the culture and perfusion of microvesselnetworks consist of a chamber holding one or more mandrels in the center(as best shown in FIG. 1). The chambers can be fabricated from anybiocompatible material and by a number of techniques, for example, bysandwiching laser-cut frames. The mandrels are assembled within thechamber in such way that they are retractable. This can be achieved byfitting the ends of the mandrels into tubing, as for example, by heatshrinking, (as demonstrated in FIG. 2). The diameter of the mandrelsdepends on the desired vessel caliber. The setup can be modified toaccommodate single vessels, two vessels, or up entire arrays of vesselsin 2D or 3D. Mandrels can be of various materials, such as polymerfibers, glass fibers, wires or the like.

Microvessels are created by seeding cells onto the mandrels, stimulatingthe cells to multiply around the mandrels, and extracting the mandrelswhen cells have formed vessel walls. The vessels are then embedded in amatrix. Depending on the culture conditions, the composition of thematrix, and the presence of angiogenic stimuli (e.g. growth factors),the parent vessels will sprout into the surrounding matrix. The sproutswill anastomoze with each other and, thus leading to the formation ofcapillary networks. After removal of the mandrels, the devices areconnected to a perfusion system, and vessels are subjected to luminalfluid flow.

Referring now to FIG. 1A, FIG. 1B and FIG. 1C, there shown is an exampleschematic of parent-vessel creation. FIG. 1A shows endothelial cells 1in a culture growth medium 100, seeded onto mandrel 2 held by shrinktubing 4 in a device body 3. FIG. 1B shows that the cells 1 havemultiplied and formed a circular layer in the form of cell-sleeve 102.FIG. 1C shows the cell-sleeve after extraction of the mandrel 2 in anextracellular matrix (ECM) gel 110 being perfused with culture growthmedium 100.

The method disclosed herein comprises the engineering of perfusablebioartificial vessel structures for tissue-engineering applications andresearch models. The general principle of the disclosed method involvesthe culture of cells in layers around removable mandrels that aretightly fit into thin-wall tubing or other fittings. Once the celllayers have reached a desired wall thickness, the mandrels are removed,and the hereby-created bioartificial vessels (BAVs) may be perfused withculture medium, blood, blood substitutes, or other fluids by aid of aperfusion system. The disclosed method allows for the production of massmanufactured or custom-created blood vessels, perfused in vitroangiogenesis models, wound healing devices, tissue components, wholetissues and organs, as well as research models.

Manufacture of Culture/Perfusion Devices

Referring now to FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D there shown is anexample schematic of a known heat-shrink process. As shown specificallyin FIG. 2A each culture/perfusion device (CPD) may comprise one or moremandrels 2 held by a supporting frame 12. The mandrels 2 of the diameterof the desired vessel caliber are fit with their ends tightly intomedical-grade shrink tubing segments 4. The mandrels 2 may comprisebiocompatible fibers (e.g. polymer, glass, wires or equivalents) havingdiameters from several micrometers up to several millimeters dependingon the vessel size being emulated. In one example, microcapillary tubingcomprising optical fibers was employed as mandrels.

As shown in the more detailed drawing of FIG. 2B, a central portion 14of each shrink tubing segment 4 is heat-shrunk around one of themandrels 2. Subsequently, as specifically shown in FIG. 2C, the mandrel2 is retracted, and the tubing cut. FIG. 2D shows the situation afterre-positioning the mandrel such that both ends of the mandrel areenclosed by the now cut-and-separated shrink tubing segment 4. Theframes 12 may be fabricated using various materials and techniques. Thesetup may be modified to accommodate either single bioartificial vesselsor arrays of bioartificial vessels. Similarly, by layering severalplanes of mandrel arrays, a thick, perfusable tissue may be generatedwith vascular networks.

Machining of Perfusion Chambers

Referring now to FIG. 3A a known setup for the perfusion of severalmandrel/shrink-tubing assemblies 11 is shown. A frame 20 mayadvantageously be milled from polycarbonate or equivalent materials.Distribution chambers 30 may be included into the design, which allowsfor simultaneous perfusion of many bioartificial vessels. Ends of a setof threads comprising the mandrels 2 are gathered in a silicon tube 23.

Laser Cutting of Mylar Frames

Referring now to FIG. 3B a novel design used in a manufacturing methodfor mounting culture/perfusion devices is schematically shown. A singlevessel design, CPD 70, may advantageously be created by sandwiching amandrel 2 held by heat-shrink tubing 4 between two laser-cut Mylar®frames 22. A cylindrical epoxy manifold 21, constructed as detailedbelow, may advantageously be used for holding the mandrel/shrink-tubingassembly 11.

Mandrel/shrink-tubing assemblies may be sandwiched between two frames ofa polyester film or the like, such as Mylar®, with adhesive sidespressed together such that each mandrel is suspended in the frame window76 by two shrink-tubing segments 4′ at each end. The two shrink-tubingsegments 4′ are stabilized and strengthened by inclusion of at least onethin stabilizing wire 26 in the frame 22 and by encapsulation incylindrical epoxy manifolds that are cast around the shrink-tubing andthe at least one thin stabilizing wire 26 by use of a mold of siliconetubing. The two shrink-tubing segments 4′ will eventually become theinflow and outflow ports for the CPD 70.

Referring now to FIG. 4A and FIG. 4B, there schematically shown is amethod for creation of manifolds for culture profusion devices. FIG. 4Aparticularly shows a plurality of shrink-tubing/mandrel assemblies 11pulled through a sleeve of, for example, silicone tubing 50. An epoxyglue 40 is injected to fill the silicone tubing 50 and allowed toharden.

FIG. 4B particularly shows the condition after the epoxy glue 40 hashardened and the silicone tubing 50 is slit open and removed. Remainingis a hardened epoxy rod 44. The epoxy rod 44 is cut after the mandrelshave been retracted behind the cutting spot leaving channels 42 createdby the shrink tubing. The ends 46 of many shrink tubes may be integratedto form a manifold 21. Stacking of individual CPDs or CPD frameassemblies can be used to create 3D vessel arrays.

Alternative Methods

Referring now to FIG. 5A, FIG. 5B and FIG. 5C there schematically shownis an alternative design for microfabricated culture/perfusion devices.FIG. 5A particularly shows a set of mandrels 2 introduced through smallperforations 54 in a frame where the perforations have sleeves 56, whichsubstitute for the shrink tubing. FIG. 5B particularly shows a CPDbefore cell seeding including a set of mandrels 2 mounted in a framewall 52.

FIG. 5C particularly shows an alternate example of a culture/perfusiondevice with vessels 62 where microfabricated manifolds 64 may beattached to the sleeves 56 on the outside of the frame 52. The vessels62 are grown on mandrels as shown herein and remain after the mandrelsare removed. Microfabrication methods, such as micro molding, may beused for the mass production of such CPD frame assemblies.

Vessel Creation and Perfusion

Referring now to FIG. 6 there schematically shown is a cell-seedingprocedure. In order to prepare the CPDs 70 for cell seeding, they arefirst cleaned and then UV-sterilized. Under sterile conditions, the CPDsare fixed to a surface, e.g. the bottom of the Petri dish 72. The innerwindow 76 (as shown in FIG. 3B) of the CPD frame assembly 70 is thenfilled with a solution that contains an attachment-protein, such aslaminin-1. One or more spacers 77 may be used as necessary. After anincubation period, the attachment-protein containing solution isremoved, and a suspension of the desired cell type (e.g. smooth musclecells or endothelial cells) in culture medium is then transferred intothe window 76 of the CPD 70.

Cell seeding may be done by filling a volume of cell suspension into thewindow, and flipping the CPD frame assembly 70 upside down, thuscreating a hanging droplet 80. During an incubation period of about 45min., a large number of cells will attach to the mandrel/shrink tubingassemblies within the CPD frame assembly. Excessive cells will sink intothe tip of the hanging drop and may be easily collected and discarded.The Petri dish, containing one or more CPD frame assemblies, is thenreturned into an upright position, filled with culture medium until theCPD frame assemblies are flooded, and incubated. The incubationconditions in one example were in an environment of 5% CO₂ at 37° C. Thecells attached to the mandrel/shrink tubing assemblies will spread outand multiply, forming concentric monolayers (e.g. endothelial cells) ormultilayers of 150 μm and more in thickness (e.g. smooth muscle cells).

At the desired wall configuration or thickness the mandrels areextracted, thereby creating hollow cellular tubes. Thinner walls may beprotected from rupture by casting a gel such as, for example, agarose,collagen, a gel of basement membrane proteins or the like, around thecell sleeves prior to mandrel extraction. The manifolds of the CPD frameassemblies are then connected to a perfusion system and perfused withthe fluid of choice, such as growth medium.

In another embodiment, a method for the creation of endothelial “parent”vessels from human vascular endothelial cells (HUVEC) comprises thesteps wherein:

-   -   The culture device is first cleaned and then sterilized by UV        exposure for 30 min. from each side. Under sterile conditions,        the device is fixed to the bottom of a Petri dish with sterile        strips.    -   The inner window of the device is then filled with an        attachment-protein solution of laminin-1 (other attachment        proteins, such as fibronectin, fibrin, or gelatin can be used        instead).    -   After overnight incubation, the attachment-protein containing        solution is removed, and a suspension of human vascular        endothelial cells in culture medium is then transferred into the        window of the device.    -   The Petri dish is then flipped upside down, thus creating a        hanging drop of cell-medium suspension in the window of the        device. After a 45 min. incubation period in a cell culture        incubator (5% CO₂, 37° C.) a large number of cells will be        attached to the mandrel/shrink tubing assemblies within the        devices.    -   The Petri dish is then brought back into the upright position,        and filled with growth medium for human vascular endothelial        cells until the device is submerged.    -   Cells not bound to the mandrels will float off and can be        aspirated and discarded.    -   The Petri dish is then placed in an incubator (5% CO₂, 37° C.).        The cells attached to the mandrels will spread out and multiply,        forming concentric monolayers of human vascular endothelial        cells.    -   The culture medium is then removed from the Petri dish. A        collagen solution is filled into the window of the culture        device, and allowed to solidify, thus embedding the mandrel with        the cell layer.    -   The human vascular endothelial cells will form sprouts into the        collagen gel. The mandrel is then slowly extracted, leaving        behind a perfusable “parent” microvessel of human vascular        endothelial cells.    -   The manifolds of the device are then connected to a perfusion        system and perfused with human vascular endothelial cells growth        medium.        Perfusion System

The CPDs may be attached to perfusion systems either in linear or incirculatory mode. A linear setup may be created with a gravity flowsystem, or a commercially available or custom-built syringe pump.Syringes are filled with perfusion medium, mounted into the syringe pumpand connected to the upstream ends of the CPDs via gas-tight tubing. TheCPDs may be stored in an incubator under sterile conditions or a sterilecell culture environment may be established within the CPD. Thedownstream manifold of the CPDs are connected to end reservoirs thatcollect the perfusate. A circulatory system may be built by using aperistaltic pump. Both, the linear and the circulatory system may befitted with devices for gas exchange. Gas concentration, perfusionpressure, flow, temperature, and the concentration of nutrients andmetabolic byproducts are measured with sensors. The collected data maybe fed into a feedback loop, allowing for tight control of the desiredparameters.

Specific Applications

Models for Angiogenesis Related Research

Referring now to FIG. 7, FIG. 7 shows a schematic of a capillary networkbetween two bioartificial parent vessels 200, 202. The fluid perfusate204 is re-routed through the capillaries 206 by decreasing the flow (f)into the “venous” parent vessel 202, and increasing the resistance (R)in the “arterial” parent vessel 200. Consequently, the perfusate 204 isdriven from the vessel with higher pressure to the vessel with lowerpressure, simulating natural blood flow from the arterial end to thevenous end of the capillary bed.

The mandrel method may be also used for the development of models forangiogenesis research, as needed for pharmaceutical testing and researchin wound repair, aging, and diseases like cancer, diabetes, arthritis,and psoriasis. Using endothelial cells only, or combinations ofendothelial cells, smooth muscle cells, and pericytes, parentbioartificial microvessels (BMVs) can be cultured around micron-diametermandrels, and embedded into a supportive gel of extracellular matrix.The mandrels will then be extracted, leaving behind patent endothelialcell tubes within the extracellular matrix gel 210. The extraction maybe done by hand, or by aid of an automated device, and with speedsvarying from extremely slow to extremely fast. Other variations mayinclude the extraction of the mandrel from bioartificial microvessels ina frozen state, coating of the mandrels with a thermo-responsivepolymer, or pulling on either end of the mandrel, and thereby thinningit until rupture.

The sprouting of the parent vessels into the surrounding gel 210 will beinduced by compounds, such as basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF), and phorbol12-myristate-13-acetate (PMA), which are added to the gel and/orperfusate (e.g. growth medium).

Complex capillary networks 222 may be created by establishing a pressuredifference between two adjacent parent bioartificial microvessels,thereby imitating arterial and venous blood flow. The fluid flow willthen be re-directed from the “arterial” bioartificial microvesselthrough the interconnected sprouts into the “venous” bioartificialmicrovessel.

The perfusate may advantageously comprise oxygenated cell growth medium,free of serum and angiogenic or angiostatic substances. In anotherexample the perfusate may be an oxygenated cell growth medium,supplemented with serum, and/or angiogenesis influencing compounds. Inyet another example embodiment the perfusate may be an oxygenatedphysiological salt solution. In another example the perfusate mayinclude oxygenated blood, blood components, or blood substitutes. In yetanother example embodiment the perfusate may not be an oxygenated, andoxygenation of the system is achieved by diffusion through the matrix.In yet another example embodiment angiogenic or angiostatic compoundsmay be added to a perfusate.

In one example embodiment, angiogenic and angiostatic compounds or thelike are added to the matrix. In yet another example embodiment cellscomprise genetically modified cells that release products into aperfusate or into the matrix. In yet another example embodiment thematrix may advantageously comprise fibrin, collagen, basement-membranematrices and gelatin. One type of useful matrix is Matrigel® gel. Inanother example embodiment the matrix may comprise agar, agarose,alginate, or silica gel.

In one example embodiment, the cells may be selected from the groupconsisting of endothelial cells, smooth muscle cells, pericytes, humancells, animal cells, plant cells, stem cells, muscle cells, liver cells,kidney cells, lung cells, skin cells, epithelial cells and geneticallymodified cells. Similarly, the matrix may be populated with cellsselected from the group consisting of endothelial cells, smooth musclecells, pericytes, human cells, animal cells, plant cells, stem cells,muscle cells, liver cells, kidney cells, lung cells, skin cells,epithelial cells and genetically modified cells, either dispersedthroughout the matrix, or locally concentrated. In some cases a fragmentof healthy or diseased tissue, such as cancer tissue is embedded intothe matrix.

Sprouting from parent vessels may be microscopically studied in vitro,in sectioned material or in whole-mount preparations. Perfusion of thebioartificial microvessels with fluorescent solutions (e.g. fluorescentdextrans) aids analysis of the sprout diameter, the patency of sproutlumens, and the degree of anastomization. 3D reconstruction of sproutmorphologies may be performed by z-axis stacking of epifluorescenceimages taken by a confocal microscope. The synthesis of a pericellularbasement-membrane matrix by sprouts 220 may be monitored in whole mountsand in histological (paraffin) sections by immunolabeling withanti-laminin and type IV collagen primary antibodies and fluorescent orperoxidase-tagged second antibodies.

In composite EC/SMC sprouts, the spatial relationships between the twocell types may be examined by labeling endothelial cells with aFITC-monoclonal antibody (MAb) to human CD31 (clone P2B1-Chemicon) orFITC-UEA 1 agglutinin—a specific marker for human endothelial cells.smooth muscle cells may be labeled with a MAb to human alpha-SM actinfollowed by RITC-anti-mouse second antibodies. Details of luminalstructure and interaction between endothelial cells and smooth musclecells may be obtained from paraffin sections labeled with theaforementioned reagents.

The described fabrication methods are the foundation for commercialmass-production of angiogenesis devices with a high repeatability. Withsuitable preservation (e.g. cryostorage), pre-grown parent vessels orwhole capillary networks could be made available to researchers inoff-the-shelf fashion.

Coronary-Artery Substitutes

For the creation of coronary-artery substitutes, mandrels with an outerdiameter selected to yield a coronary artery substitute having a vessellumen with an inner diameter of approximately 4 mm to 5.5 mm.Alternatively, the mandrel may be a hollow tube that is perfused andpermeable enough to allow for exchange of nutrients and gases during thegrowth period of the coronary-artery substitute. The coronary-arterysubstitutes may be grown either solely from smooth muscle cells, thuspresenting a structure analog to the media layer in blood vessels, ormade as composite structures from two or three cell types.

Smooth muscle cells are seeded onto the mandrels and grown to circularlayers of 300-400 μm. In order to speed up the creation ofcoronary-artery substitutes, the SMC-phenotype may be manipulated insuch way that the cells are brought into a highly proliferativephenotype during the initial growth phase, and then switched to adifferentiated state after the vessel wall has reached the desiredthickness. The phenotype switch will cause the smooth muscle cell's todramatically slow down their growth rate, and induce the production ofextracellular matrix proteins, such as collagen and elastin, whichaffect mechanical properties of the vessels. The phenotype switch may beachieved by controlling the expression of certain genes. For example,with aid of a tetracycline-responsive promoter, gene expression (e.g.for elastin) may be suppressed until the vessel wall has reached thedesired thickness. Omitting tetracycline from the growth medium willthen activate the inserted gene. Over-expression of elastin, forinstance, will inhibit further cell proliferation and exert structuraland signaling functions within the vessel wall. Mechanical conditioning,e.g. pulsatile flow may be used to strengthen the coronary-arterysubstitutes, and induce physiological alignment of the cells. Otherexternal or internal “phenotype switches” may be potentially used, aswell.

Endothelial cells may be seeded into the SMC sleeves either directlyafter removal of the mandrel, or after the conditioning andrestructuring of the smooth muscle cells. Endothelial cell seeding maybe done by infusion of an endothelial cell suspension into the SMCsleeve. The flow is then stopped for a period of time to allow properattachment of the endothelial cells. If necessary, the vessels may berotated, or repeatedly flipped upside down in order to facilitate aneven distribution of the endothelial cells.

Alternatively, endothelial cells may be seeded onto the mandrel first.In that case smooth muscle cells are seeded onto a confluent endothelialcell layer. For this method, it will be necessary to prevent theendothelial cells from migration towards the periphery of thecoronary-artery substitute, which is richer in oxygen and nutrients.

If desired, seeding fibroblast cells onto the outside of the SMC sleevescan create an adventitial layer.

The cells for creating coronary-artery substitutes may be derived fromautologous, heterologous, or xenogeneic material. The cells may be stemcells, precursor cells, or differentiated cells. The cells may begenetically modified to achieve a specific phenotype or to lower theimmune response of the host organism.

The herein-disclosed CPD method provides the option for mass-producingoff-the-shelf vessel substitutes, or vessel substitutes that are customdesigned for the recipient. The herein-disclosed CPD method is alsosuitable for the development of models for tissue engineering ofcoronary-artery substitutes, for research in atherogenesis,arteriogenesis, for research in the interaction of different vascularcell types with each other and with extracellular matrix components, forstudies on the effects of nitric oxide, and for the study of variespharmaceuticals.

Blood and Lymphatic Vessels of Different Size or Type

The herein-disclosed CPD method may be used to create blood vessels indiameter and type other than coronary arteries. Changing the diameter ofthe mandrel will vary the vessel diameter. The type of the vessel(arterial, venous, lymphatic) may be varied with the phenotype of thecells, and/or the time point when the proliferation of the cells isinhibited. Veins, for example, contain only a small smooth muscle celllayer.

Other Tubular-Like Tissues

The herein-disclosed CPD method may be used for the engineering of othertubular tissues, such as bile duct, lacrimal duct, pharyngotympany tube,oviduct, vas deferens, ureter, urethra, pulmonary airways etc. Theherein-disclosed CPD method may also prove useful for the generation ofnerve conduits from different cell types, including glial cells, forguidance of neural growth and repair.

BAV Systems for Engineered Tissues

The herein-disclosed CPD method may be used for the engineering oftissues and organs by using arrays of removable mandrels as scaffold.The cells of the desired tissue/organ (muscle, liver, kidney, lung,skin, etc.) are seeded onto the attachment-protein coated mandrels.These mandrels may be made from solid fibers or wires, or, alternativelyfrom perfusable permeable tubes, such cellulose. The mandrels areseparated from each other in a precise spacing that allows the singlecell sleeves to merge. With this method, sheets or blocks of tissue maybe formed. The mandrels are then extracted (or differently removed), andthe bioartificial tissue is internally perfused by aid of a perfusionsystem.

Wound Healing Device

Pre-manufactured bioartificial vessel systems may be used to assist inwound healing, such as for chronic ulcers in diabetic patients.Bioartificial capillary networks could be embedded into patches ofsupportive materials (e.g. from extracellular matrix gels, enriched withangiogenic growth factors), and placed onto the wound. Autonomouslyperfused with oxygenized nutrient solutions, the bioartificial vesselwould facilitate the sprouting of the donor vasculature and skin.Alternatively, such a bioartificial vessel patch could be sandwichedbetween the wound and a skin graft, and facilitate the in-growth of thegraft.

Gene-Therapy Device

Bioartificial vessels could be used for implantable drug deliverydevices. Cells, taken from a patient, could be genetically modified invitro to produce a certain protein (hormone, enzyme etc.). These cellsmay be then grown into bioartificial vessels or vascular networks, usingthe aforementioned method. Re-implanted into the host, the cellscontinue to produce the target substance and release it locally orsystemically.

Artificial Tissues and Organs

Tissue engineered vascular networks, as described above, may be used forthe creation of tissues, or even whole organs. One approach is thecreation of one or more in vitro perfused parent vessels. Stroma cellsfrom the desired tissue or organ are seeded around the parent vessels,as for example, in a gel. The stroma cells are supplied with nutrientsand oxygen via the parent vessels. When the stroma cells multiply, thedemand for nutrients and oxygen increases. The cells release angiogenicfactors, and stimulate the vessels to sprout. The vessel system sproutsin the same rate, as the tissue grows—very similar to the naturalgrowth. Therefore, this system would be also a good model for studies indevelopmental biology.

Another approach utilizes parallel arrays of mandrels as a scaffold forstroma cells. As the stroma cells multiply, cell layers are formedaround the mandrels. Eventually the space between all the mandrels isfilled with stroma cells, resulting in a sheet of tissue. Upon removalof the mandrels, the tissue may be perfused through the channels, leftbehind by the mandrels. Those channels can become endothelializedthrough luminal seeding. The approach is not limited to 2D. Eitherseveral sheets may be stacked, or 3D scaffolds may be used. The inventorherein has used 2D arrays as well as 3D arrays for the engineering ofmuscle tissue.

In yet another approach, layers of tissue and layers of vascularnetworks could be created independently, and then intermittentlystacked. All these approaches can produce either simple models with oneor two cell types, or rather complex constructs composed of several celltypes.

Upon implantation, the tissues or organs, engineered with these methodscould be either connected directly to the blood stream, or kept perfusedby a perfusion system until the host vasculature has grown into thegraft.

Example of Perfused Tissue Engineered Muscle Construct

Referring now to FIG. 8a , an in vitro image of an example of aplurality of mandrels after seeding with smooth muscle cells is shown. Aplurality of mandrel-and-shrink tubing units M were sandwiched on aMylar® frame. The distance between the mandrels M was adjusted toapproximately 100 μm. The ends of all shrink tubing segments werecombined in one upstream and one downstream manifold (not shown). TheMylar frame was sterilized, laminin coated and seeded with a suspensionof 5×10⁶ rat aortic smooth muscle cells SM (RASMCs)/ml. The cells SMattached to each individual mandrel M and multiplied, thus formingcircular layers. After 10 days, the individual layers had merged andresulted in one thick sheet or plate of smooth muscle cells. Afteradditional 7 days in growth medium, the medium was supplemented with 50U/ml heparin for another 7 days. Then, all mandrels were extracted, andthe tissue perfused with heparin-medium at a rate of 10 ml/day. Theperfusion chamber was kept fixed to the bottom of a 100-mm Petri dishfilled with heparin-medium. The SMC plate was perfused for 11 days. Overthat time, the channels CH remained functional and remained clearlyvisible in vitro (as best shown in FIG. 8b ).

Referring now to FIG. 8b , an example of a perfused muscle plate MP isshown. Fluid is shown perfused through the tubing ends (T) into channels(CH) left behind by the extracted mandrels.

Referring now to FIG. 9, an alternate embodiment of a CPD isschematically shown. In one example, a CPD 900 includes a layer 902juxtaposed between a first glass slide 904 and a second glass slide 920.The layer 902 has a thickness suitable for embedding a plurality offluid ports connected by channels 922. The plurality of fluid portsinclude a cell suspension port 914, a plurality of inlet ports 912 and aplurality of outlet ports 918. Ports that are connected by channels 922to allow for passage and sharing of fluids function similarly. Multipleports, such as ports 912, are arranged to provide multiple accesspoints. Other applications may advantageously employ microfluidicdesigns with fluid chambers and ports created in microfluidic materials.Similarly, the layer may comprise silicone or other materials suitablefor use in microscopy or microfluidic applications. A collagen chamber906 is advantageously located for access by a pair of hollow, flexiblecapillary tubes 916. The number of capillary tubes employed may rangefrom one to substantially more than two as may be accommodated by thesize of the CPD and the number of vessels being created. Each of theplurality of ports and chambers may be accessed through the layer by oneor more syringe pumps through tubing inlets 940A, 940B, where thesyringe pumps are attached to syringes having needles. While only twosyringe pump tube inlets 940A, 940B are shown to simplify the drawings,it will be understood that separate syringe pumps, syringes orequivalents may be used for injecting or extracting materials into eachchamber and/or port as the case may be. In one embodiment, the syringepumps are coupled to gas tight syringes.

Having described the features of the alternate embodiment CPD 900, itwill aid the understanding of the invention to now describe one methodfor constructing the CPD. In one example employing a silicone layer forlayer 902, a pattern of holes and channels is punched into a siliconelayer covered with an adhesive top layer 943 and adhesive bottom layer945. Then, hollow needles are punctured through the silicone, which arethen used to guide polyimide-coated fused-silica capillaries 916 intothe collagen chamber 906 and also into one of the inlet ports 912. Thetwo capillary tubes are held by small-bore tubing 910, leading from themain chamber into the outlet ports 908. The silicone layer 902 is thensandwiched in between two glass slides with aid of the adhesive layers.The CPD 900 is then autoclaved and stored until use. To get the chamber906 ready for vessel creation, a collagen solution is prepared, injectedthrough a syringe needle directly into the collagen chamber 906, andallowed to gel in an incubator overnight. The CPD 900 is then connectedto a syringe pump by injecting syringe needles into the two inlet ports.

The syringe needles are, in turn, connected to gas-tight tubing, whichleads to two gas-tight syringes, filled with grow medium with welladjusted pH, and mounted into a syringe pump. The two outlet ports 908A,908B are connected to waste reservoirs in similar fashion. The syringepumps, here operating as perfusion pumps, are then turned on, therebyfilling the inlet ports and sequentially priming the inlet ports, thecapillary tubes, and the outlet ports. When all the air is pushed out ofthe system, then each capillary tube is grabbed with tweezers and theends that reach into the collagen chamber are pulled back through thecollagen gel until only the ends of the capillaries reach into thematrix chamber. With this procedure, two perfusable channels are createdin the collagen gel. In order to seed cells into the collagen channels,a highly concentrated suspension of endothelial cells is injected intothe ports for cell suspension. The syringe pump is then turned off, andthe other ends of the capillaries are then pulled back into the smallreservoirs 914R that contain the cells, leading to an immediate influxof large numbers of cells into the collagen channels. The flow rate ofthe cells can be tightly controlled through the height of the wastereservoirs. The CPD is then placed in an incubator for 45 min. forallowing the cells to attach to the walls of the collagen channels. TheCPD can be flipped over several times or otherwise manipulated todistribute the cells optimally. Finally, the capillary tubes are pulledout of the cell reservoirs into reservoirs that are part of the inletport, and the syringe pump is turned on and set to the desired perfusionrate. Excessive cells are washed out. This seeding procedure leads totwo parent vessels with homogeneous monolayers of cells after allowingtime for growth, where the time required is shorter for more highlyconcentrated numbers of cells injected into the tubes. Note that themandrel may be removed from the matrix by extraction and/ordecomposition, depending on the type of mandrel used.

Referring now to FIG. 10, a single parent vessel 1050 growing sprouts1052 into a surrounding matrix 1054 is shown. When Human Umbilical CordVein Cells (HUVECs) are seeded into the collagen channels, the socreated parent vessels begin to sprout into the collagen. These sproutselongate and begin to branch. These branches eventually anastomoze withbranches from the opposite parent vessel and, thus, form vascularnetworks.

Referring now to FIG. 11, a first parent vessel 1102 is shown connectedthrough a network of sprouts 1106 to a second parent vessel 1104. Thesprouts 1106 have lumens and are perfused.

Protocol for Creation of Parent Vessels

Referring now to FIG. 12, an alternate method for creating parent cellsby seeding channels in a collagen matrix is shown. Having described thealternate CPD using a silicone layer, a specific example of anapplication for creating a microvessel system will now be described tofacilitate understanding of the disclosure by those skilled in the art.

The CPD is sterilized in an autoclave, and kept in a sterile environmentuntil use. A collagen solution is prepared and kept on ice. The collagenis filled into a small syringe. The syringe is fitted with a 30G syringeneedle, and the collagen solution is injected into the collagen chamberthrough the syringe needle until the chamber is completely filled withcollagen 1002. A second syringe needle is injected from the oppositeside of the chamber as an air outlet.

The CPD is then connected to a syringe pump by injecting syringe needlesinto the two inlet ports 1004. The syringe needles are, in turn,connected to gas-tight tubing, which leads to two gas-tight syringes,filled with grow medium with well adjusted pH, and mounted into thesyringe pump. The two outlet ports are connected to waste containers insimilar fashion (i.e. syringe needles injected into the outlet ports,with tubing leading to the waste containers) 1006.

The CPD is perfused by operating the syringe pump as a perfusion pump,thereby filling the inlet ports and sequentially priming the inletports, the capillary tubes, and the outlet ports 1008. When all the airis pushed out of the system (e.g. through small diameter syringe needlesserving as removable air outlets), then each capillary tube is grabbedwith tweezers and the ends that reach into the collagen chamber arepulled back through the collagen gel until only the ends of thecapillaries reach into the chamber. With this procedure, two perfusablechannels are created in the collagen gel 1010.

In order to seed cells into the collagen channels, a highly concentratedsuspension of endothelial cells is injected into the ports for cellsuspension 1012. The syringe pump is then turned off, and the other endsof the capillary tubes are then pulled back from the inlet ports intothe small reservoirs that contain the cells, leading to an immediateinflux of large numbers of cells through the capillary tubes into thecollagen channels 1014. The flow rate of the cells can be tightlycontrolled through the backpressure (height of the waste reservoirs).The capillaries are then pulled back further into the reservoirs thatare connected to the inlet ports.

The CPD is then placed in an incubator for 45 min for allowing the cellsto attach to the walls of the collagen channels 1016. The CPD can beflipped over several times or otherwise manipulated to distribute thecells optimally.

Finally, the syringe pump is turned on and set to a desired perfusionrate 1020. Excessive cells are washed out. This seeding procedure leadsto two parent vessels with homogeneous monolayers of cells 1022. One ormore microvessel networks may be created by perfusing the parent vesselsas described above.

Alternately the procedure for creating the parent vessels may alsoinclude embedding mandrels into the collagen matrix, extracting themandrels, and infusing cells into the channels left behind by themandrels as well as seeding cells onto mandrels as described above withreference to FIGS. 1A-1C and others. The combination of the two methodsallows layering of different cell types.

The invention has been described herein in considerable detail in orderto comply with the Patent Statutes and to provide those skilled in theart with the information needed to apply the novel principles of thepresent invention, and to construct and use such exemplary andspecialized components as are required. However, it is to be understoodthat the invention may be carried out by specifically differentequipment, and devices and reconstruction algorithms, and that variousmodifications, both as to the equipment details and operatingprocedures, may be accomplished without departing from the true spiritand scope of the present invention.

The complete disclosures of all references cited in this specificationare hereby incorporated by reference. In the event of an otherwiseirreconcilable conflict, however, the present specification shallcontrol.

REFERENCES

-   Akhtar N, Dickerson E B, Auerbach R. 2002. The sponge/Matrigel    angiogenesis assay. Angiogenesis 5:75-80.-   Algire G H, Chalkley H W, Legallais F Y, Park H D. 1945. Vascular    reactions of normal and malignant tissues in vivo. I. Vascular    reactions of mice to wounds and to normal and neoplastic    transplants. J Natl Cancer Inst 6:73-85.-   Andrade S P, Machado R D, Teixeira A S, Belo A V, Tarso A M, Beraldo    W T. 1997. Sponge-induced angiogenesis in mice and the    pharmacological reactivity of the neovasculature quantitated by a    fluorimetric method. Microvasc Res 54:253-261.-   Arthur W T, Vernon R B, Sage E H, Reed M J. 1998. Growth factors    reverse the impaired sprouting of microvessels from aged mice.    Microvasc Res 55:260-270.-   Ausprunk D H, Knighton D R, Folkman J. 1974. Differentiation of    vascular endothelium in the chick chorioallantois: a structural and    autoradiographic study. Dev Biol 38:237-248.-   Clark E R, Clark E L. 1939. Microscopic observations on the growth    of blood capillaries in the living mammal. Am J Anat 64:251-301.-   Davis G E, Camarillo C W. 1996. An alpha 2 beta 1 integrin-dependent    pinocytic mechanism involving intracellular vacuole formation and    coalescence regulates capillary lumen and tube formation in    three-dimensional collagen matrix. Exp Cell Res 224:39-51.-   Elsdale T, Bard J. 1972. Collagen substrata for studies on cell    behavior. J Cell Biol 54:626-637.-   Feder J, Marasa J C, Olander J V. 1983. The formation of    capillary-like tubes by calf aortic endothelial cells grown in    vitro. J Cell Physiol 116:1-6.-   Fishman J A, Ryan G B, Karnovsky M J. 1975. Endothelial regeneration    in the rat carotid artery and the significance of endothelial    denudation in the pathogenesis of myointimal thickening. Lab Invest    32:339-351.-   Folkman J, Haudenschild C. 1980. Angiogenesis in vitro. Nature    288:551-556.-   Folkman J, Haudenschild C C, Zetter B R. 1979. Long-term culture of    capillary endothelial cells. Proc Natl Acad Sci U S A 76:5217-5221.-   Gimbrone M A, Jr. 1976. Culture of vascular endothelium. In: Prog    Hemost Thromb. p 1-28.-   Gimbrone M A, Jr., Cotran R S, Folkman J. 1974a. Human vascular    endothelial cells in culture. Growth and DNA synthesis. J Cell Biol    60:673-684.-   Gimbrone M A, Jr., Cotran R S, Leapman S B, Folkman J. 1974b. Tumor    growth and neovascularization: an experimental model using the    rabbit cornea. J Natl Cancer Inst 52:413-427.-   Greenblatt M, Shubi P. 1968. Tumor angiogenesis: transfilter    diffusion studies in the hamster by the transparent chamber    technique. J Natl Cancer Inst 41:111-124.-   Hoying J B, Boswell C A, Williams S K. 1996. Angiogenic potential of    microvessel fragments established in three-dimensional collagen    gels. In Vitro Cell Dev Biol Anim 32:409-419.-   Jaffe E A, Nachman R L, Becker C G, Minick C R. 1973. Culture of    human endothelial cells derived from umbilical veins. Identification    by morphologic and immunologic criteria. J Clin Invest 52:2745-2756.-   Jozaki K, Marucha P T, Despins A W, Kreutzer D L. 1990. An in vitro    model of cell migration: evaluation of vascular endothelial cell    migration. Anal Biochem 190:39-47.-   Koike T, Vernon R B, Gooden M D, Sadoun E, Reed M J. 2003. Inhibited    angiogenesis in aging: a role for TIMP-2. J Gerontol A Biol Sci Med    Sci 58:B798-805.-   Kubota Y, Kleinman H K, Martin G R, Lawley T J. 1988. Role of    laminin and basement membrane in the morphological differentiation    of human endothelial cells into capillary-like structures. J Cell    Biol 107:1589-1598.-   Kuzuya M, Kinsella J L. 1994. Induction of endothelial cell    differentiation in vitro by fibroblast-derived soluble factors. Exp    Cell Res 215:310-318.-   Maciag T, Kadish J, Wilkins L, Stemerman M B, Weinstein R. 1982.    Organizational behavior of human umbilical vein endothelial cells. J    Cell Biol 94:511-520.-   Madri J A. 1982. Endothelial cell-matrix interactions in hemostasis.    Prog Hemost Thromb 6:1-24.-   Madri J A, Pratt B M. 1986. Endothelial cell-matrix interactions: in    vitro models of angiogenesis. J Histochem Cytochem 34:85-91.-   Madri J A, Pratt B M, Tucker A M. 1988. Phenotypic modulation of    endothelial cells by transforming growth factor-beta depends upon    the composition and organization of the extracellular matrix. J Cell    Biol 106:1375-1384.-   Madri J A, Stenn K S. 1982. Aortic endothelial cell migration. I.    Matrix requirements and composition. Am J Pathol 106:180-186.-   Manoussaki D, Lubkin S R, Vernon R B, Murray J D. 1996. A mechanical    model for the formation of vascular networks in vitro. Acta Biotheor    44:271-282.-   Marx M, Perlmutter R A, Madri J A. 1994. Modulation of    platelet-derived growth factor receptor expression in microvascular    endothelial cells during in vitro angiogenesis. J Clin Invest    93:131-139.-   Merwin J R, Anderson J M, Kocher O, Van Itallie C M, Madri    J A. 1990. Transforming growth factor beta 1 modulates extracellular    matrix organization and cell-cell junctional complex formation    during in vitro angiogenesis. J Cell Physiol 142:117-128.-   Montesano R, Orci L. 1985. Tumor-promoting phorbol esters induce    angiogenesis in vitro. Cell 42:469-477.-   Montesano R, Orci L, Vassalli P. 1983. In vitro rapid organization    of endothelial cells into capillary-like networks is promoted by    collagen matrices. J Cell Biol 97:1648-1652.-   Montesano R, Pepper M S, Orci L. 1993. Paracrine induction of    angiogenesis in vitro by Swiss 3T3 fibroblasts. J Cell Sci 105 (Pt    4):1013-1024.-   Mori M, Sadahira Y, Kawasaki S, Hayashi T, Notohara K, Awai M. 1988.    Capillary growth from reversed rat aortic segments cultured in    collagen gel. Acta Pathol Jpn 38:1503-1512.-   Nehls V, Drenckhahn D. 1995. A novel, microcarrier-based in vitro    assay for rapid and reliable quantification of three-dimensional    cell migration and angiogenesis. Microvasc Res 50:311-322.-   Nehls V, Herrmann R. 1996. The configuration of fibrin clots    determines capillary morphogenesis and endothelial cell migration.    Microvasc Res 51:347-364.-   Neumann T, Nicholson B S, Sanders J E. 2003. Tissue engineering of    perfused microvessels. Microvasc Res 66:59-67.-   Nicosia R F, Bonanno E, Smith M, Yurchenco P. 1994a. Modulation of    angiogenesis in vitro by laminin-entactin complex. Dev Biol    164:197-206.-   Nicosia R F, Bonanno E, Villaschi S. 1992. Large-vessel endothelium    switches to a microvascular phenotype during angiogenesis in    collagen gel culture of rat aorta. Atherosclerosis 95:191-199.-   Nicosia R F, Nicosia S V, Smith M. 1994b. Vascular endothelial    growth factor, platelet-derived growth factor, and insulin-like    growth factor-1 promote rat aortic angiogenesis in vitro. Am J    Pathol 145:1023-1029.-   Nicosia R F, Ottinetti A. 1990. Modulation of microvascular growth    and morphogenesis by reconstituted basement membrane gel in    three-dimensional cultures of rat aorta: a comparative study of    angiogenesis in matrigel, collagen, fibrin, and plasma clot. In    Vitro Cell Dev Biol 26:119-128.-   Nicosia R F, Tchao R, Leighton J. 1982. Histotypic angiogenesis in    vitro: light microscopic, ultrastructural, and radioautographic    studies. In Vitro 18:538-549.-   Nicosia R F, Tchao R, Leighton J. 1983. Angiogenesis-dependent tumor    spread in reinforced fibrin clot culture. Cancer Res 43:2159-2166.-   Nicosia R F, Tuszynski G P. 1994. Matrix-bound thrombospondin    promotes angiogenesis in vitro. J Cell Biol 124:183-193.-   Passaniti A, Taylor R M, Pili R, Guo Y, Long P V, Haney J A, Pauly R    R, Grant D S, Martin G R. 1992. A simple, quantitative method for    assessing angiogenesis and antiangiogenic agents using reconstituted    basement membrane, heparin, and fibroblast growth factor. Lab Invest    67:519-528.-   Pepper M S, Montesano R, Vassalli J D, Orci L. 1991. Chondrocytes    inhibit endothelial sprout formation in vitro: evidence for    involvement of a transforming growth factor-beta. J Cell Physiol    146:170-179.-   Rosen E M, Meromsky L, Setter E, Vinter D W, Goldberg I D. 1990.    Quantitation of cytokine-stimulated migration of endothelium and    epithelium by a new assay using microcarrier beads. Exp Cell Res    186:22-31.-   Sage E H, Vernon R B. 1994. Regulation of angiogenesis by    extracellular matrix: the growth and the glue. J Hypertens Suppl    12:S145-152.-   Vernon R B, Angello J C, Iruela-Arispe M L, Lane T F, Sage    E H. 1992. Reorganization of basement membrane matrices by cellular    traction promotes the formation of cellular networks in vitro. Lab    Invest 66:536-547.-   Vernon R B, Gooden M D. 2002. New technologies in vitro for analysis    of cell movement on or within collagen gels. Matrix Biol 21:661-669.-   Vernon R B, Lara S L, Drake C J, Iruela-Arispe M L, Angello J C,    Little C D, Wight T N, Sage E H. 1995. Organized type I collagen    influences endothelial patterns during “spontaneous angiogenesis in    vitro”: planar cultures as models of vascular development. In Vitro    Cell Dev Biol Anim 31:120-131.-   Vernon R B, Sage E H. 1999. A novel, quantitative model for study of    endothelial cell migration and sprout formation within    three-dimensional collagen matrices. Microvasc Res 57:118-133.-   Villaschi S, Nicosia R F. 1993. Angiogenic role of endogenous basic    fibroblast growth factor released by rat aorta after injury. Am J    Pathol 143:181-190.

What is claimed is:
 1. A device for creating perfusable vessels in vitrocomprising: a device body including a silicone layer sandwiched betweena first layer and a second layer; a plurality of fluid ports embedded inthe silicone layer; a plurality of port connecting channels embedded inthe silicone layer; at least one cell reservoir port embedded in thesilicone layer, the cell reservoir port being connected by a first portconnecting channel to an inlet port and connected by a second portconnecting channel to an outlet port; a matrix chamber containingcollagen embedded in the silicone layer, the matrix chamber having oneor more perfusion channels formed in the collagen; a plurality ofnon-permeable capillary tubes each coupled at one end to one or more ofthe plurality of fluid ports and having a second end; wherein only thesecond end of each non-permeable capillary tube reaches into the matrixchamber; and wherein the first end of each non-permeable capillary tubeis located to be pulled back into the at least one cell reservoir port.2. The device of claim 1 further comprising a perfusion system connectedto a pair of said inlet and outlet ports which are individuallyconnected to opposing ends of at least one of the plurality of portconnecting channels.
 3. The device of claim 2 where the perfusion systemcomprises at least one syringe pump.
 4. The device of claim 2 where theperfusion system comprises at least one peristaltic pump.
 5. The deviceof claim 2 the perfusion system comprises a gravity flow system.
 6. Thedevice of claim 1 wherein the plurality of non-permeable capillary tubescomprise polyimide-coated fused-silica capillaries.